The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
In specific binding assays, such as, e.g., immunoassays, DNA hybridization assays, receptor-binding assays, and cellular binding assays, generally the analytes to be measured are present at very low concentrations. Therefore, various labelling compounds have been developed that allow the labelled reactant to be detected and quantitated at high sensitivity. In immunoassays and DNA hybridization assays time-resolved luminescence spectroscopy using lanthanide chelates is well known (e.g., I. Hemmila, T. Stahlberg, and P. Mottram (eds.), "Bioanalytical Applications of Labelling Technologies", Wallac, Turku, 1994). Stable photoluminescent (referred in the context of this specification simply as luminescent) lanthanide chelates also have other applications, e.g. fluorescence microscopy and cytometry. Therefore, a number of attempts have been made to develop new highly luminescent chelate labels suitable for those types of time-resolved fluorometric applications. These include, e.g., stable chelates composed of derivatives of pyridines (U.S. Pat. No. 4,920,195, U.S. Pat. No. 4,801,722, U.S. Pat. No. 4,761,481, PCT WO FI-91/00373, U.S. patent application Ser. No. 08/135,525, now U.S. Pat. No. 5,459,186,Remuinan, M. J., Roman, H., Alonso, M. T. and Rodriguez-Ubis, J. C., 1993, J. Chem. Soc., Perkin Trans.2, 1099), bipyridines (U.S. Pat. No. 5,216,134), terpyridines (U.S. Pat. No. 4,859,777, U.S. Pat. No. 5,202,423, U.S. Pat. No. 5,234,825) or various phenolic compounds (U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191, Ital. Pat. 42508 A/89) as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives (U.S. Pat. No. 5,032,677, U.S. Pat. No. 5,055,578, U.S. Pat. No. 4,772,563), macrocyclic cryptates (U.S. Pat. No. 4,927,923, PCT WO 93/5049, EP-A493,745) and macrocyclic Schiff bases (EP-A369,000) have been patented. None of these terbium chelates fulfill all the required features to be used as labels for bioaffinity assays. These requirements are high thermodynamic and kinetic chelate stability, hydrophilicity, high absorptivity at a suitable excitation wavelength, appropriate triplet state to enable efficient energy transfer, high luminescence intensity, presence of a functional group, allowing the formation of a covalent linkage between the chelate and the target molecule, and the retention of the affinity and nonspecific binding properties of the used biomolecules. In case of Tb.sup.III, the energy gap between the excitation state of the donor ligand and the emitting level of the Tb.sup.III ion should be high enough to prevent energy back transfer (Sabbatini, N., Mecati, A., Guardigli, M., Balzani, V., Lehn, J.-M., Zeissel, R. and Ungaro, R., 1991, J. Luminescence 48 & 49; 463-468). Moreover, with exactly the same ligand structure, none of the labels made from the prior art chelates has enough high luminescence intensities with both Eu.sup.III and Tb.sup.III. In some applications, which contain various chromatographic separation steps, it would be preferable that the label structures differ from each other only with respect to the lanthanide used. Besides, the same key intermediates can be used in the preparations of several lanthanide labels, e.g., Eu.sup.III and Tb.sup.III labels.